Milk fermenting product

ABSTRACT

A method of producing a mixed bacterial concentrate which comprises separately incubating in separate culture media two or three types of bacteria, the first type being selected from the group consisting of Streptococcus lactis, Streptococcus cremoris, Lactobacillus bulgaricus and Streptococcus thermophilus, the second type being selected from the group consisting of Streptococcus citrovorus and Streptococcus paracitrovorus, and the third type consisting of Streptococcus diacetylactis, concentrating the respective media to obtain separate concentrates of the two or three type of bacteria, mixing together the two or three types of bacteria in the desired proportions to produce a mixed concentrate without permitting further growth of the bacteria, and then freezing the mixed concentrate so that it can be stored for a long time without major loss in the viability of the bacteria. A stabilized mixed bacteria concentrate consisting essentially of a substantially neutralized mixture of two or three types of bacteria, as aforesaid, the concentrate being stabilized by the admixture of a stabilizing agent and a nutrient medium and the concentrate being frozen so that it can be stored for a long period of time without major loss in the viability of the bacteria.

United States Patent [1 1 Farr [11] E Re. 28,488

[ Reissued July 22, 1975 i 1 MILK FERMENTING PRODUCT [75] Inventor:Stewart M. Farr, Sarasota, Fla.

[73] Assignee: Microlife Technics, lnc., Sarasota,

Fla.

Reissue of:

[64] Patent No.: Re. 28.276

Issued: Dec. 17, 1974 Appl. No.: 417,134 Filed: Nov. 19, 1973 Which ls aReissue of:

[64] Patent No.: 3,420,742

Issued: Jan. 7, 1969 Appl. No.: 404,526 Filed: Oct. 16, 1964 US.Applications:

[63] (ontinuationJn-part of Ser. No. 285.858. June 6.

1963.2ihandoned.

OTHER PUBLlCATlONS Lamprcch (thesis), 62-4703, Production and Storage ofDairy Starter Organisms, Univ. of Wisconsin, May

Longworth ct al., Journal of Bacteriology, Vol. 29, No. 6, pp. 595607(1935).

Hargrove, Journal of Dairy Science, 1961, Vol. 44, pp. 1799 to 1810.

Lamprech et al., Journal of Applied Bacteriology, Vol. 26, No. 3,December 1963, pp. 359369.

Journal of Dairy Science, Vol. 45, pp. 1263 to 1266 and 1290 to 1294,October 1962.

Primary Examiner-Lionel M. Shapiro Attorney, Agent, or FirmMiller,Morriss, Pappas & McLeod [57] ABSTRACT A method of producing a mixedbacterial concentrate which comprises separately incubating in separateculture media two or three types of bacteria, the first type beingselected from the group consisting of Streptococcus lactis,Streptococcus cremoris, Lactobacillus bulgaricus and Streptococcusthermophilus, the second type being selected from the group consistingof Streptococcus citrovorus and Streptococcus paraci trovorus, and thethird type consisting of Streptococcus diacetylactis, concentrating therespective media to obtain separate concentrates of the two or threetype of bacteria, mixing together the two or three types of bacteria inthe desired proportions to produce a mixed concentrate withoutpermitting further growth of the bacteria, and then freezing the mixedconcentrate so that it can be stored for a long time without major lossin the viability of the bacteria. A stabilized mixed bacteriaconcentrate consisting essentially of a substantially neutralizedmixture of two or three types of bacteria, as aforesaid, the concentratebeing stabilized by the admixture of a stabilizing agent and a nutrientmedium and the concentrate being frozen so that it can be stored for along period of time without major loss in the viability of the bacteria.

9 Claims, No Drawings 1 4 MILK FERMENTING PRODUCT Matter enclosed inheavy brackets! ,Iappears in the original patent but forms no part ofthe first and this reissue specification; matter printed in italicsindicates the additions made by the first reissue. Matter enclosed indouble heavy brackets [[nappears in the first reissue patent but formsno part of this reissue specification; matter printed ln bold faceindicates the additions made by this reissue.

[[This application is a continuation-in-part of my co-pendingapplication Ser. No. 285.858. filedJune 6. I 963, now abandoned.]]

This is a divisional reissue application of reissue application Ser. No.417.134. filed Nov. 19. 1973. now Reissue Patent No. Re. 28.276.reissued Dec. 17. 1974 of Original N0. 3.420.742 and is acontinuation-in-part of application Ser. No. 285.858. filed June 6.1963. now abandoned.

This invention relates to a concentrated bacterial product useful forfermenting milk in order to make cottage cheese, cheese, butter,cultured buttermilk and other cultured milk products, and also relatesto a process for producing said bacterial product.

in the following description and claims, the term .acid-producingbacteria" shall refer to bacteria capable of fermenting lactose orother, similar carbohydrates rapidly at temperatures of about 70 degreesFahrenheit to l degrees Fahrenheit with the production of principallylactic acid, namely, Streptococcus lactis, Streptococcus cremoris,Lactobacillus bulgaricus and Streptococcus thermophilus. The termflavorproducing bacteria shall referto bacteria capable of fermentingcitric acid or citrates at a favorable pH with the production ofbiacetyl, actylmethylcarbinol, volatile acids and carbon dioxide.namely, Leuconostoc citrovorum (Streptococcus citrovorus) andLeuconostoc dextranicum (Streptococcus paracitrovorus) and subspeciesthereof. Streptococcus diacetylactis (a species not recognized inBergeys Manual. Breed, Murray and -Hi'tchins, i957) produces somelactic, acid and also produces substantial amounts of flavorconstituents.

Cottage cheese, cheese, butter and cultured buttermilk are manufacturedby a fermenting procedure using a suitable bacteria. Dairies usuallypurchase an initial culture of a mixture of suitable bacteria from acommercial organization supplying same. This culture is then propagatedin the dairy by placing the culture in milk and incubating it at asuitable temperature, usually'at around 70 degrees Fahrenheit. for asuitable period of time. usually from l4 to 18 hours. in order to form amother culture. The propagation step is repeated a number of times usingthe bacterial product of the preceding step as the inoculum and finallythe propagation is carried out in a rather large quantity of milk inorder either to produce a bulk starter, which can then be used forfermenting the final batch of milk to make the end product, or toproduce cultured buttermilk.

if sufficient care is not taken both in the manufacture of the motherculture and in the propagation thereof, bacterioph'agemay become presentin the bulk starter. or perhaps earlier in the propagation process, andmay proliferate so as to hinder the growth of or completely destroy thebacteria. This may cause delayed setting or no setting of the milk. ifthis contamination occurs,

large quantities of milk are often lost and all of the time and laborspent in propagating the culture are wasted. Contamination of theinitial culture is not a serious problem because the initial culture isproduced under carefully controlled conditions with special equipmentand by skilled personnel. However, few dairies and the like. where mostof the propagating is presently performed, are so equipped. Thus, theincidence of such contamination during the propagation of the bacteriaaccording to present practices in dairies is believed to be higher thannecessary.

The initial culture most usually acquired from a supplier consists of amixture of at least two of the three types of bacteria referred .toabove, namely, acidproducing bacteria and flavor-producing bacteria andStreptococcus diacetylactis. The types of bacteria must be properlybalanced in order to produce acceptable products and the properbacterial balance for producing one product, such as cottage cheese, isdifferent than the proper balance required for another product,

such as cultured buttermilk. The types of bacteria do not grow equallywell in a propagation process of the type described above because plainmilk favors the growth of the acid-producing bacteria at the expense ofthe flavor-producing bacteria. Thus, even though the mother culture mayhave the proper balance of the types of bacteria, there may be a seriousunbalance by the time the bulk starter has been made. Moreover, thisunbalance may not be discovered until an unacceptable end product hasbeen produced, which also results in major economic losses.

it is possible to overcome these problems by suitable care and controlover the propagating process, but to do so requires constant supervisionby a skilled person having a sound knowledge of dairy bacteriology, andit also requires a considerable investment in equipment.

Moreover, because of the time required to produce the bulk starter,dairies must anticipate their need for a cultured milk product severaldays in advance. Thus, they may be unable to supply an unexpected demandfor a particular product on short notice. Also they may find that by thetime the bulk starter is made the anticipated demand for the culturedmilk product may not have materialized and the bulk starter may have tobe thrown away, all of which involves considerable loss.

in addition, even under optimum conditions the present starter culturesare bulky and have a relatively short life. They must be used soon afterthey are made and they are not easy to store. For example, in making aslow set cottage cheese approximately eight (8) quarts of starter arerequired to inoculate one hundred (I00) gallons of milk; while in a fastset process, which is-the one commonly used, approximately five timesthis amount of 40 quarts of inoculum are required. When it is realizedthat it is customary to inoculate at least 10 times this amount of milkor 1,000 gallons at one time. it will be seen that the preparing,storage and handling of the inoculum becomes costly and, because of thenecessity of maintaining bacteria viability, it is a difficult problem.

Accordingly, a need exists for a bacterial product for use by dairieswhich is free of bacteriophage and which contains acid-producingbacteria and flavor-producing bacteria or acid-producing bacteria.flavor-producing bacteria and Streptococcus diacetylactis in the properbalance, which is of such high potency or concentration that amplesupplies are easy to store, and which is prepared in such a manner thatit will remain viable .in 'a stored condition for an extended period oftime.This bacterial product can be placed directly .into milk forforming the end product, without further propagation inthe dairy, sothat the cultured end product can be produced rapidly and inexpensively.The present invention is intended to meet the need for such product."

Further, there are certain types of culttired dairy products. ofwhichyoghurt is an example. for which the demand is low so that they eitherare not produced at 1 bacteriophage free, stabilized and concentratedbac,te-

rial product consisting of a mixture of acid-producing bacteria andflavor-producing bacteria or a mixture of acid-producing bacteria,flavor-producing bacteria and Streptococcus diacetylactis having a hightotal bacterial concentration, about 5 cubic centimeters .of the.

concentrate containing approximately the same number of bacteria as arepresent in one quart of. fullylfermerited milk or, in other words, aconcentrate whose bacteria count is approximately 189 times the bacteriacount in fully fermented milk. Otherwise stated, the bacterialconcentrate has l0-6OX cells per ml. The bacterial product contains byadmixture at least about the mixture is frozen toproduce the stabilized,concentrated, bacterial product of the invention.

The 'three' types of bacteria are grown in culture "media in "accordancewith conventional practice. The

particular procedures used for growing the three types of bacteriacan'bewidely varied and form no part of the present invention. However, thebacteria must be grownseparately in separate culture media and care mustbe take'n'to insurethat the bacteria are phagefree. Suitable proceduresare known to the dairy industry for maintaining phage-free conditions inbacterial cultures. In general, such procedures involve carefulselection of the initial culture, the provision of a suitablecu'ltui'erotation system and the maintenance of proper standards of cleanlinessand sterilization in the equipmentand' the area in' which the culturingoperation is carried out.

) A preferred procedure for culturing the acidprodu cin g bacteriaand,,also,"Streptococcus diacetylacti' 's, so as to preventbacteriophage development without hindering culture activity, isdescribed in the Journal' of Dairy Science." October I96]. volume XLlV.N0. 1( pages l7 99 i810 and U.S. 'Pat. N0. 3.04l.248.

2 percent by weight of glycerol plus a suitable nutrient mediumand isquickly frozen whereby the viability of the bacteria is maintained at ahigh level for a long time period. The ratio of the acid-producingbacteria to the flavor-producing bacteria or v the ratio of theacidproducing bacteria to the flavor-producing bacteria and theStreptococcus diacetylactis is carefully controlled and is adjusted asneeded in order to adapt the bacterial product to the production of aparticular type of cultured milk product. In general, the percentagefofthe flavor-producing bacteria concentrate plus the percentage of theStreptococcus diacetylactis concentrate ranges from about 2 to about 12percent of the total count and the acid-producing bacteria concentrateconstitutes the balance of the end product.

According to the method aspects of the invention, it is a criticalfeature of the invention that the acidproducing bacteria, theflavonproducing bacteria and the Streptococcus diacetylactis be grownand concentrated separately from each other. The three types of bacteriaare separately grown or propagated in their respective culture media andthen they are concentrated by centrifuging to produce separateconcentrated baccounts but, in general, each ml, of the concentrateswill contain Ill-60X lll cells per ml. The two or three types ofconcentrated bacteria are then mixed with each other in the properproportions depending on usage and with glycerol and with a nutrientmedium and then r Specifically. a culture medium is made by dissolving 5pounds of'powdei'ed ski m'milk. 10 pounds of lactose and 2 /2 pounds ofBasamin Busch yeast in 60 gallons of water which has been preheated toI30 degrees Fahrenheit. 3.4 pounds of NagHPOa H20 and 5.1'

pounds of KH POQ are dissolved in distilled water and are added to andmixed in the culture medium. The medium is heated to 200 degreesFahrenheit and held at that temperature for V2 hour. 1.5 pounds ofK4P2O7 are dissolved in distilled water and thissolution is added to theculture mediumheld at 200 degrees Fahrenheit. The solution is.cooled;t o72 degrees Fahrenheit and then it is inoculated with a mother culture ofthe acidproducing bacteria or. Streptococcus diacetylactis after whichit is incubated for, from l8 to ZOhours at from 72- degreesFahren-heit,to 7.7 degrees Fahrenheit.

, It may be necessary to neutralize the culture medium for theacid-producing bacteria during the culturing step by addingsuitableneutralizing agents at selected times. Ordinarily, theneutralizing agent is added when the pH of the medium drops to 5.2 andsufficient neutralizing agent is added to raise the'pH to about 6.6.

- ing the acid-producing bacteria while avoiding the development ofphage so that the product of this culturing step is especiallysatisfactory for the purposes of this invention.

The preferred procedure for culturing the flavorproducing bacteria isthe same as that used for the acid- -producing bacteria -except thattheculture medium may be different and, for example, may be trypsin digestmilk, cottage cheesewhey or corn steep plus dextros e (whichispreferredybecause these latter media favor thegrowt'h of theflavor-producing bacteria more than does a medium consisting essentiallyof plain milk.

With the flavor-producing bacteria, it usually is not necessary toneutralize the culture medium except when corn steep is used as theculture medium. In all other respects, the procedure for culturing theflavorproducing bacteria can be the same as that previously described"respect to the acid-producing bacteria and Streptococcus diacetylactisand, hence, needs no further description.

After the three types of bacteria have been separately grown as abovedescribed, each is separated from the medium in which it has-been grownand is concentrated by passing the medium containing the bacteriathrough a suitable centrifugal apparatus so that the liquid isdischarged from the apparatus and the bacteria collect on the bowl ofthe apparatus from whence they may be scraped out or mechanicallyejected during the operation of the centrifuge. The three types ofbacteria are separately centrifuged so that separate concentrates of thethree types of bacteria are obtained. Apparatus capable of centrifugingculture mediums to separate and fresh culture medium and then 60 ouncesof glycerol were added to the mixture so that glycerol amounted to aboutpercent by weight of the total concentrate. However. as little as about2 percent by weight of glycerol can be used as a minimum while percentor more by weight of glycerol can be used, if desired. The

higher percentages of glycerol are not needed to maintain the viabilityo f'the bacteria, but they can effect other advantages which make theuse of such amounts desirable in some instances. For example, use ofabout 20 percent or more by weight of glycerol provides a. 1

concentrate which is especially well suited for making yoghurt. Such aconcentrate remains liquid below 32 degrees Fahrenheit and may,therefore, be more convenient for home use. I

After mixing the glycerol and nutrient medium, them two or threeindividual bacteria concentrates are then mixed together in suitableproportions to form a mixed bacterial product which is concentrated andphagefree. The amounts of the two or three individual bacteriaconcentrates which'are mixed together will depend upon the particularfermented product to be formed by the mixed bacterial product. Where themixed bacterial product is to be used for making buttermilk. about l-4percent of the total bacteria of the mixed concentrate should beStreptococcus diacetylactis, about 8 percentshould be flavor-producingbacteria and the balance should be acid-producing bacteria. Wherecottage cheese or hard cheeses are to be made. about 98 percent of thetotal bacteria in the mixed bacterial concentrate should beacid-producing bacteria and about 2 percent should be flavor-producingbacteria. One or the other of the flavor-producing bacteria andStreptococcus diacetylactis can be eliminated from the bacterialconcentrate for producing cottage cheese, depending on tasterequirements.

Where the mixed bacterial concentrate is to be stored for anyappreciable period of time, i.e., more than a week or so, it should befrozen after it is formed and, preferably be held at about 4 degreesFahrenheit because in such condition the viability of the bacteria willbe maintained for a long time.

A suitable amount of the concentrated and mixed bacterial product can bemixed with milk. either fresh,

whole milk, skim milk or reconstituted dry milk, so that 'the milk canbe fermented thereby in order to form a fermented milk product. Theconcentrated, mixed bacterial product can be placed directly into themilk to be processed without any intermediate 'propagations. In otherwords, the concentrated, mixed bacterial product of the invention servesin place of the bulk starter conventionally used for this purpose. Inall other respects, the method of fermenting the milk to form the endproduct can be in accordance with conventional procedures. The highbacteria concentration in the mixed bacterial concentrate of theinvention make it possible, however, to prepare fermented milk productsin a much shorter period of time than is required according toconventional procedures, simply by using larger amounts of theconcentrate. Specifically, by adding rel- ,atively large amounts of theconcentrate to milk, it is possible to reduce the time of setting themilk to an hour or so. In this fashion, it possible for a consumer .tomake certain types of cultured milk products, such as yoghurt, in thehome. Also, a dairy can use this technique to produce small quantitiesof cultured milk products rapidly. v

Furthermore, this invention eliminates the troublesome problem,especially for small operators, of anticipating the demand for certaincultured milk products, which is presently necessary,'several days inadvance when he must start the propagating sequence. That is, he cankeep a supply of phage-free, concentrated ,mixed bacterial product onhand for use on relatively short notice in precise quantities, so thatthe desired end product can be produced rapidly, usually on the same daythat the need for same becomes apparent.

Example I. Preparation of Cottage Cheese I00 gallons of milk werepasteurized by heating to i 200 degrees Fahrenheit for about a half-houror longer.

The milk was cooled to degrees Fahrenheit and then 60 ccs. of themixedbacterial concentrate containing about 98 percent by weight ofacid-producing bacteria and about 2 percent by weight offlavor-producing bacteria, was added to it and thoroughly mixed. Themixture was incubated at 78 degrees Fahrenheit for about 14 hours andthere was obtained a cottage cheese product of pleasing flavor andproper consistency.

Example II. Preparation of Cultured Buttermilk 100 gallons of milk werepasteurized by heating to 200 degrees Fahrenheit and holding for about ahalf hour. The milk was cooled to 74 degrees Fahrenheit and wasinoculated with one ounce of the "bacterial concentrate containing aboutpercent by weightof acid-producing bacteria and about 8 percent byweight of flavor-producing bacteria and 2 percent of Streptococcusdiacetylactis. The mixture was incubated for approximately 14 hours at74 degrees Fahrenheit until its titratable acidity expressed as lacticacid reached about 0.85 to 1.0 percent. Themixture was then cooled andagitated to a smooth consistency to produce a cultured buttermilkproduct.

\ ounces of instant powdered milk and one cubic centi meter of the mixedbacterial concentrate containing 20 percent weight of glycerol weremixed together and then allowed to stand at 74 degrees Fahrenheit for 24hours until thickened. The yoghurt product has a pleasing flavor and thetypical consistency of yoghurt preparations. By using larger amounts ofthe concentrate or faster growing bacteria, such as L. bulgaricus or byincubating at a higher temperature the time for incubation can bereduced to as little as one hour or so.

Although particular, preferred embodiments of the invention have beendisclosed herein for illustrative purposes, it will be understood thatvariations or modiflcations of such disclosure, which lie within thescope of the appended claims are fully contemplated.

What is claimed is;

[[1. The method of producing a mixed bacterial concentrate useful forculturing milk which comprises:

(a) separately incubating in separate culture media two types ofbacteria, the first type being selected from the group consisting ofStreptococcus lactis, Streptococcus cremoris, Lactobacillus bulgaricusand Streptococcus thermophilus which produce lactic acid wherein theincubation culture medium contains skim milk and wherein during theincubation the lactic acid produced is neutralized as the pH of themedium drops by adding a neutralizing agent to maintain the growth ofthe bacteria, and the second type being selected from the groupconsisting of Streptococcus citrovorus and Streptococcus paracitrovorus,wherein the culture medium contains skim milk or a digest milk toproduce bacteria which are centrifugable from the media to form aconcentrate;

(b) concentrating the respective media to obtain separate concentratesof the two bacteria [,1 containing at least about l l0 cells/ml. each;

(c) mixing together the two types of bacteria in the desired proportionsto produce a mixed concentrate having closely controlled amounts of thetwo types of bacteria therein without permitting further growth of thebacteria; and

(d) then freezing the mixed concentrate so that it can be stored for along time without major loss in the viability of the bacteria.]]

[[2. The method of claim 1, including the steps of mixing glycerol and anutrient medium in the concentrates in precise relative proportions.]]

[[3. The method of claim 1, including the step of neutralizing theculture medium for said first type of bacteria to maintain the pHthereof between about 5.2 and about 6.6.]]

4. A stabilized, mixed bacteria concentrate containing at least about10X 10 cells/ml each consisting essentially of a substantiallyneutralized mixture of two types of bacteria which have been separatelyincubated, concentrated and then mixed, the first type being selectedfrom the group consisting of Streptococcus lacs tis, Streptococcuscremoris, Lactobacillus bulgaricus and Streptococcus thermophilus whichhas been incubated in a culture medium containing skim milk so as toproduce lactic acid and wherein during the incubation the lactic acidproduced has been neutralized with a neutralizing agent to maintain thegrowth of the bacteria,

and the second type being selected from the group consisting ofStreptococcus citrovorus and Streptococcus paracitrovorus which has beenincubated in skim milk or a digest milk, said bacteria having beenseparated from the media and concentrated in a centrifuge. saidconcentrate being stabilized by the admixture of a stabilizing agent anda nutrient medium so that the concentrate is stabilized against rapidloss of viability. said concentrate being frozen so that it can bestored for a long period of time without major loss in the viability ofthe bacteria.

Claim 4 was deleted in Re. 28,276, but is reinstated in this reissuewith amendments.

[5. A stabilized mixed bacteria concentrate according to claim 4, inwhich the concentrate contains at least about 10X 10 cell per ml. 1

6. A stabilized mixed bacteria concentrate according to claim 4.. inwhich the stabilizing agent is glycerol.

Claim 6 was deleted in Re. 28.276. but is reinstated in this reissue.

7. A stabilized mixed bacteria concentrate according to claim 4, inwhich the bacteria of said first type comprise of at least about 88% ofthe total count in the bacteria concentrate.

Claim 7 was deleted in Re. 28,276, but is reinstated in this reissue.

[8. The method of producing a mixed'bacteria] concentrate whichcomprises separately incubating in separate culture media three types ofbacteria, the first type being selected from the group consisting ofStreptococcus lactis, Streptococcus cremoris, Lactobacillus bulgaricusand Streptococcus thermophilus, the second type being selected from thegroup consisting of Streptococcus citrovorus and Streptococcusparacitrovorus, and the third type consisting of Streptococcusdiacetylactis, concentrating the respective media to obtain separateconcentrates of the three bacteria, mixing together the three types'ofbacteria in the desired proportions to produce a mixed concentratehaving closely controlled amounts of the three types of bacteria thereinwithout permitting further growth of the bacteria, and then freezing themixed concentrate so it can be stored for a long time without major lossin the viability of the bacteria] [9. .The method of claim 8, includingthe steps of mixing glycerol and a nutrient medium in the concentratesin precise relative proportions] 10. A stabilized, mixed bacteriaconcentrate containing at least about 10 10 cells/m] each consistingessentiallyof a substantially neutralized mixture of three types ofbacteria which have been separately incubated, concentrated and thenmixed, the first type being selected from the group consisting ofStreptococcus lac tis, Streptococcus cremoris, Lactobacillus bulgaricusand Streptococcus thennophilus which has been incubated in a culturemedium containing skim milk so as to produce lactic acid and whereinduring the incubation the lactic acid produced has been neutralized witha neutralizing agent to maintain the growth of the bacteria, the secondtype being selected from the group consisting of Streptococcuscitrovorus and Streptococcus paracitrovorus which has been incubated inskim milk or in a digest milk and the third type consisting ofStreptococcus diacetylactis, said bacteria having been separated fromthe media and concentrated in a centrifuge, said concentrate beingstabilized by the admixture of a stabilizing agent and a nutrient mediumso that the concentrate is stabilized against rapid loss of viability,said concentrate being frozen so that it can be stored for a long periodof time without major loss in the viability of the bacteria.

Claim 10 was deleted in Re. 28,276. but is reinstated in this reissuewith amendments.

Claim 12 was deleted in Re. 28,276, but is reinstated in this reissue.

13. A stabilized mixed bacteria concentrate according to claim 10, inwhich the bacteria of the second type comprise about 8% ot'the totalcount ofthe bacteria concentrate, the bacteria of the third typecomprise between about 1% and 4% of the total count of the bacteriaconcentrate. the balance being bacteria of the first type.

Claim 13 was deleted in Re. 28,276, but is reinstated in this reissue.

14. A stabilized bacteria concentrate consisting essentially of asubstantially neutralized concentrate of a [[bacteriaII bacterium whichis separately incubated and concentrated selected from the groupconsisting of Streptococcus citrovorus and Streptococcus paracitrovoruswhich has been incubated in a culture medium containing skim milk or adigest milk, said bacteria having been separated from the media andconcentrated in a centrifuge. said concentrate being stabilized by theadmixture of a stabilizing agent and a nutrient medium so that theconcentrate is stabilized against rapid loss of viability, theconcentrate being frozen so it can be stored for a long time withoutmajor loss in the viability of the bacteria, the concentrate containingat least about lOX cells per ml.

Claim [4 was deleted in Re. 28,276, but is reinstated in this reissuewith amendments. I

15. A stabilized. mixed bacteria concentrate containing at least about10X [0 cells/ml each consisting esincubated, concentrated and thenmixed, the first type consisting of Streptococcus lactis which has beenincubated in a culture medium containing skim milk and wherein duringthe incubation the lactic acid produced has been neutralized with aneutralizing agent to maintain the growth of the bacteria, the secondtype being selected from the group consisting of Streptococcuscitrovorus and Streptococcus paracitrovorus which has been incubated inskim milk or a digest milk, and the third type consisting ofStreptococcus diacetylactis which has been incubated and neutralized inthe manner of the first type of bacteria, said bacteria having beenseparated from the media and concentrated in a centrii'ug'e. saidconcentrate being stabilized by the admixture of a stabilizing agent anda nutrient medium so that the concentrate is stabilized against rapidloss of viability,

said concentrate being frozen so that it can be stored for a long periodof time without major loss in the via- 7 bility of the bacteria.

16. A stabilized, mixed bacteria concentrate containing at least about10X 10 cells/ml each consisting essentially of a substantiallyneutralized mixture of three types of bacteria which have beenseparately incubated, concentrated and then mixed, the first typeconsisting of Streptococcus lactis, which has been incubated in aculture medium containing skim milk and which has been neutralized witha neutralizing agent during incubation to maintain the growth of thebacteria, the second type being selected from the group consisting ofStreptococcus citrovorus and Streptococcus paracitrovorus which has beenincubated in a culture medium containing skim milk or a digest milk, andthe third type consisting of Streptococcus diacetylactis which has beenincubated and neutralized in the manner of the first type of bacteria,said bacteria having been separated from the media and concentrated in acentrifuge, said concentrate being stabilized by the admixture of astabilizing agent and a nutrient medium so that the concentrate isstabilized against rapid loss of viability, said concentrate beingfrozen so that it can be stored for a long period of time without majorloss in the viability of the bacteria.

Claim 16 was deleted in Re. 28,276, but is reinstated in this reissuewith amendments.

[[17. The method of producing a mixed bacterial concentrate which comprises:

(a) separately incubating in separate culture media three types ofbacteria, the first type being selected from the group consisting ofStreptococcus lactis, Streptococcus cremoris, Lactobacillus bulgaricusand Streptococcus thermophilus, which produce lactic acid wherein theincubation culture medium contains skim milk and wherein duringincubation the lactic acid produced is neutralized as the pH of themedium drops by adding a neutralizing agent to maintain the growth ofthe bacteria, the second type being selected from the group consistingof Streptococcus citrovorus and Streptococcus paracitrovorus wherein theculture media contains skim milk or a digest milk, and the third typeconsisting of Streptococcus diacetylactis which is incubated andneutralized in the manner of the first type of bacteria to producebacteria which are centrifiigable from the media to form a concentrate;

(b) concentrating the respective media to obtain separate concentratesof the three bacteria containing at least about 1O l0 cells/ml. each;

(c) mixing together the three types of bacteria in the desiredproportions to produce a mixed concentrate having closely controlledamounts of the three types of bacteria therein without permittingfizrther growth of the bacteria; and

(d) then freezing the mixed concentrate so it can be stored fora longtime without major loss in the viability of the bacterial] [[18. Themethod ofcloim 17, including the steps of mixing glycerol and a nutrientmedium in the concentrates in precise relative proportions.]]

4. A STABILIZED, MIXED BACTERIA CONCENTRATE CONTAINING AT LEAST ABOUT 10X 10**9 CELLS/ML EACH CONSISTING ESSENTIALLY OF A SUBSTANTIALLYNEUTRALIZED MIXTURE OF TWO TYPES OF BACTERIA WHICH HAVE BEEN SEPARATELYINCUBATED, CONCENTRATED AND THEN MIXED, THE FIRST TYPE BEING SELECTEDFROM THE GROUP CONSISTING OF STREPTOCOCCUS LACTIS, STREPTOCOCCUSCREMORIS, LACTOBACILLUS BULGARICUS AND STREPTOCOCCUS THERMOPHILUS WHICHHAS BEEN INCUBATED IN A CULTURE MEDIUM CONTAINING SKIM MILK SO AS TOPRODUCE LACTIC ACID AND WHEREIN DURING THE INCUBATION THE LACTIC ACIDPRODUCED HAS BEEN NEUTRALIZED WITH A NEUTRALIZING AGENT TO MAINTAIN THEGROWTH OF THE BACTERIA, AND THE SECOND TYPE BEING SELECTED FROM THEGROUP CONSISTING OF STREPTOCOCCUS CITROVORUS AND STREPTOCOCCUSPARACITROVORUS WHICH HAS BEEN INCUBATED IN SKIM MILK OR A DIGEST MILK,SAID BACTERIA HAVING BEEN SEPARATED FROM THE MEDIA AND CONCENTRATED IN ACENTRIFUGE, SAID CONCENTRATE BEING STABLIZED BY THE ADMIXTURE OF ASTABLIZING AGENT AND A NUTRIENT MEDIUM SO THAT THE CONCENTRATE ISSTABLIZED AGAINST RAPID LOSS OF VIABILITY, SAID CONCENTRATE BEING FROZENSO THAT IT CAN BE STORED FOR A LONG PERIOD OF TIME WITHOUT MAJOR LOSS INTHE VIABILITY OF THE BACTERIA.
 6. A stabilized mixed bacteriaconcentrate according to claim 4, in which the stabilizing agent isglycerol. -------1 Claim 6 was deleted in Re. 28,276, but is reinstatedin this reissue.
 7. A stabilized mixed bacteria concentrate according toclaim 4, in which the bacteria of said first type comprise of at leastabout 88% of the tOtal count in the bacteria concentrate. -------1 Claim7 was deleted in Re. 28,276, but is reinstated in this reissue.
 10. Astabilized, mixed bacteria concentrate containing at least about 10 X109 cells/ml each consisting essentially of a substantially neutralizedmixture of three types of bacteria which have been separately incubated,concentrated and then mixed , the first type being selected from thegroup consisting of Streptococcus lactis, Streptococcus cremoris,Lactobacillus bulgaricus and Streptococcus thermophilus which has beenincubated in a culture medium containing skim milk so as to producelactic acid and wherein during the incubation the lactic acid producedhas been neutralized with a neutralizing agent to maintain the growth ofthe bacteria , the second type being selected from the group consistingof Streptococcus citrovorus and Streptococcus paracitrovorus which hasbeen incubated in skim milk or in a digest milk and the third typeconsisting of Streptococcus diacetylactis, said bacteria having beenseparated from the media and concentrated in a centrifuge, saidconcentrate being stabilized by the admixture of a stabilizing agent anda nutrient medium so that the concentrate is stabilized against rapidloss of viability, said concentrate being frozen so that it can bestored for a long period of time without major loss in the viability ofthe bacteria. -------1 Claim 10 was deleted in Re. 28,276, but isreinstated in this reissue with amendments.
 12. A stabilized mixedbacteria concentrate according to claim 10, in which the stabilizingagent is glycerol. -------1 Claim 12 was deleted in Re. 28,276, but isreinstated in this reissue.
 13. A stabilized mixed bacteria concentrateaccording to claim 10, in which the bacteria of the second type compriseabout 8% of the total count of the bacteria concentrate, the bacteria ofthe third type comprise between about 1% and 4% of the total count ofthe bacteria concentrate, the balance being bacteria of the first type.-------1 Claim 13 was deleted in Re. 28,276, but is reinstated in thisreissue.
 14. A stabilized bacteria concentrate consisting essentially ofa substantially neutralized concentrate of a ((bacteria)) bacteriumwhich is separately incubated and concentrated selected from the groupconsisting of Streptococcus citrovorus and Streptococcus paracitrovoruswhich Has been incubated in a culture medium containing skim milk or adigest milk, said bacteria having been separated from the media andconcentrated in a centrifuge said concentrate being stabilized by theadmixture of a stabilizing agent and a nutrient medium so that theconcentrate is stabilized against rapid loss of viability, theconcentrate being frozen so it can be stored for a long time withoutmajor loss in the viability of the bacteria, the concentrate containingat least about 10 X 109 cells per ml. -------1 Claim 14 was deleted inRe. 28,276, but is reinstated in this reissue with amendments.
 15. Astabilized, mixed bacteria concentrate containing at least about 10 X109 cells/ml each consisting essentially of a substantially neutralizedmixture of three (two) types of bacteria which have been separatelyincubated, concentrated and then mixed , the first type consisting ofStreptococcus lactis which has been incubated in a culture mediumcontaining skim milk and wherein during the incubation the lactic acidproduced has been neutralized with a neutralizing agent to maintain thegrowth of the bacteria , the second type being selected from the groupconsisting of Streptococcus citrovorus and Streptococcus paracitrovoruswhich has been incubated in skim milk or a digest milk, and the thirdtype consisting of Streptococcus diacetylactis which has been incubatedand neutralized in the manner of the first type of bacteria, saidbacteria having been separated from the media and concentrated in acentrifuge said concentrate being stabilized by the admixture of astabilizing agent and a nutrient medium so that the concentrate isstabilized against rapid loss of viability, said concentrate beingfrozen so that it can be stored for a long period of time without majorloss in the viability of the bacteria. -------1 Claim 15 was deleted inRe. 28,276, but is reinstated in this reissue with amendments.
 16. Astabilized, mixed bacteria concentrate containing at least about 10 X109 cells/ml each consisting essentially of a substantially neutralizedmixture of three types of bacteria which have been separately incubated,concentrated and then mixed , the first type consisting of Streptococcuslactis, which has been incubated in a culture medium containing skimmilk and which has been neutralized with a neutralizing agent duringincubation to maintain the growth of the bacteria, the second type beingselected from the group consisting of Streptococcus citrovorus andStreptococcus paracitrovorus which has been incubated in a culturemedium containing skim milk or a digest milk, and the third typeconsisting of Streptococcus diacetylactis which has been incubated andneutralized in the manner of the first type of bacteria, said bacteriahaving been separated from the media and concentrated in a centrifuge,said concentrate being stabilized by the admixture of a stabilizingagent and a nutrient medium so that the concentrate is stabilizedagainst rapid loss of viability, said concentrate being frozen so thatit can be stored for a long period of time without major loss in theviability of the bacteria. -------1 Claim 16 was deleted in Re. 28,276,but is reinstated in this reissue with amendments. ((17. The method ofproducing a mixed bacterial concentrate which comprises: (a) separatelyincubating in separate culture media three types of bacteria, the firsttype being selected from the group consisting of Streptococcus lactis,Streptococcus cremoris, Lactobacillus bulgaricus and Streptococcusthermophilus, which produce lactic acid wherein the incubation culturemedium contains skim milk and whereIn during incubation the lactic acidproduced is neutralized as the pH of the medium drops by adding aneutralizing agent to maintain the growth of the bacteria, the secondtype being selected from the group consisting of Streptococcuscitrovorus and Streptococcus paracitrovorus wherein the culture mediacontains skim milk or a digest milk, and the third type consisting ofStreptococcus diacetylactis which is incubated and neutralized in themanner of the first type of bacteria to produce bacteria which arecentrifugable from the media to form a concentrate; (b) concentratingthe respective media to obtain separate concentrates of the threebacteria containing at least about 10 X 109 cells/ml. each; (c) mixingtogether the three types of bacteria in the desired proportions toproduce a mixed concentrate having closely controlled amounts of thethree types of bacteria therein without permitting further growth of thebacteria; and (d) then freezing the mixed concentrate so it can bestored for a long time without major loss in the viability of thebacteria.)) ((18. The method of claim 17, including the steps of mixingglycerol and a nutrient medium in the concentrates in precise relativeproportions.))